1. Technical Field
The present invention relates to a novel composition having a built-in mechanism that utilizes citrate, Ca.sup.2+ and HPO.sub.4.sup.-2 in a defined ratio and a specific protocol capable of preventing autoclave-elicited precipitation and caramelization in the manufacturing process. The composition is particularly useful as a rich energy source for ocular tissues under surgery, with concurrent suppression of lactic acid formation and accumulation in the cells. It may be used in surgeries in general, although other uses, for example, tissue storage and preparation for grafting, and topical application are also contemplated.
2. Background Information
The present invention is distinctly different from the prior art (Chen, et al., U.S. Pat. No. 5,116,868). Specifically, it contains no glucose and has a built-in mechanism that utilizes citrate, Ca.sup.2+ and HPO.sub.4.sup.-2 in a defined ratio, together with a specific protocol that includes heating the solution under vacuum, followed with rapid cooling, to prevent autoclave-elicited precipitation.
Sterilization is an essential element in the manufacturing process. Heat sterilization after closing (sealing the container) is preferred by the pharmaceutical industry for safety reasons. Filtration, which proceeds before closing, is considered risky although it is acceptable to the Food and Drug Administration for sterilizing solutions that contain heat-labile components.
Filtration as a sterilization means was used in the prior art (Chen, et al., U.S. Pat. No. 5,116,868) because the medium contains 5.5 mM glucose, pH 7.4, which is known to caramelize when heated. In addition, however, it is discovered that autoclave manifests not only caramelization, but also precipitation. These problems have rendered the composition unacceptable to the pharmaceutical industry despite its efficacy.
The source of precipitation was traced to calcium salts, and a mechanism means was discovered to overcome the precipitation problem. It is indispensable for solutions containing HPO.sub.4.sup.-2 and Ca.sup.+2 to be heat sterilized. It is also useful for manufacturing solutions that contain 5.5 mM glucose at pH 7.4 without caramelization. The rationale for the latter is not fully understood, but is probably attributable to the O.sub.2 -free environment.
The discovery of a means to prevent autoclave-elicited precipitation and caramelization in the manufacturing process is a significant scientific as well as technological achievement. There was no hint in prior arts as to how to overcome these problems. Prior to applicant's invention, in order to autoclave, Ca.sup.2+ and HPO.sub.4.sup.-2 have to be separated and glucose had to be present in high concentrations, about 5%-10% or 275 mM-550 mM, at acidic pH (around 5.0). These conditions are non-physiological accordingly harmful to tissues. For instance, in the methods of Garabedian, et al. (U.S. Pat. Nos. 4,443,432 and 4,550,022), the ophthalmic irrigating solution (trade name BSS-plus, marketed by Alcon, Fort Worth, Tex.) is manufactured by separating Ca.sup.2+ from phosphate and NaHCO.sub.3 in two bottles and by using filtration to sterilize the small bottle that contains 5.1 mM glucose.
The present use of citrate is distinctly different from the prior art (Chen, et al., U.S. Pat. No. 5,116,868), where citrate is employed as an antioxidant for protecting .beta.-hydroxybutyrate from oxidation because the solution is not O.sub.2 -free. Because of this use, citrate can be replaced by one or more of antioxidants such as glutathione, vitamin E and ascorbate. Whereas, in this novel mechanism, citrate is added to prevent autoclave-elicited precipitation, and citrate can be replaced by isocitrate, another tricarboxylic acid, but not antioxidants.
The complete effect of citrate is not fully understood. Solubility of calcium salts is not a sole determining factor, because di- and mono-carboxylic acids such as oxaloacetate, maleate, fumerate, succinate, malate, and acetate form more soluble calcium salts, but are not as effective (see Table 1). These acids have to be present in about 10-20 times higher concentrations and even then are only effective to prevent precipitation up to 90.degree. C.
The composition of the prior art contains 10 mM citrate, 20 mM acetate, 2 mM Ca.sup.2+, 5 mM HPO.sub.4.sup.-2, and 5mM H.sub.2 PO.sub.4.sup.-1. Table 1 shows that Ca.sup.2+ salts of these anions, except HPO.sub.4.sup.-2, are within soluble ranges. The [Ca.sup.2+ ][HPO.sub.4.sup.-2 ] value (10.0) is greater than the solubility product at 38.degree. C. ([Ca.sup.2+ ][HPO.sub.4.sup.-2 ]=(1.8).sup.2 =3.24) and less than that at 100.degree. C. (19.36). According to the solubility theory, the solution should precipitate (as CaHPO.sub.4) at room temperature, but clear at .gtoreq.100.degree. C. However, since it is clear at room temperature, the level of free Ca.sup.2+ must has been reduced to &lt;0.65 mM through chelation with anions. Occurrence of precipitation at elevated temperatures is thus inconsistent with both the solubility and free Ca.sup.2+ theories, and is therefore totally unexpected.
TABLE 1 ______________________________________ Solubility of Selected Calcium Salts Anions Solubility (mM) ______________________________________ H.sub.2 PO.sub.4.sup.-1 71.4 (30.degree. C.) HPO.sub.4 -2 1.8 (38.degree. C.), 4.4 (100.degree. C.) Acetate 2364.5 (0.degree. C.), 1877.7 (100.degree. C.) Citrate 14.9 (18.degree. C.), 16.8 (23.degree. C.) Fumarate 101.4 (30.degree. C.) Malate 39.0 (0.degree. C.), 58.8 (37.5.degree. C.) Maleate 167.9 (25.degree. C.), 186.5 (40.degree. C.) Malonate 20.5 (0.degree. C.), 33.6 (100.degree. C.) Succinate 9.1 (10.degree. C.), 41.9 (80.degree. C.) ______________________________________ Re-calculated from data in Handbook of Chemistry and Physics, R. C. Weast editor, 49th ed., the Chemical Rubber Co., Cleveland, OH, pp. B185-B-188, 1968.
Glucose, a major energy source for mammalian cells, is used in the irrigating solutions of prior arts to mimic in vivo conditions. However, it is discovered that under surgical conditions the cells of vascularized tissues continue to extract glucose from the blood circulation; whereas, those of avascular tissues use endogenous glucose from glycogen (a glucose storage in vivo) when glucose is absent in the irrigating solution. In addition, in the presence of .beta.-hydroxybutyrate, glucose metabolism, and thus lactate production, are reduced. Duration of endogenous glucose availability is thus extended, which ranges from several hours to several days depending on glycogen storage levels. Therefore, exogenous .beta.-hydroxybutyrate is adequate to serve as an energy source for tissues to sustain their viability and to perform physiologic functions in ocular and other short term surgeries.
With the novel mechanism, the present composition is able to combine with many other components for specific applications, such as the additions of glucose for application in lengthy surgery and perfusion if desired, of polymers to form a slow-release drug delivery system, and of essential components selected from Medium 199 for optimal corneal storage.
Utilization of the viscous solution as a slow-release drug delivery vehicle is an unique and novel concept. Diffusion rate of solutes in viscous solutions is markedly reduced and the high viscosity will extend the duration of the solution's contact with tissues. When drugs such as antibiotics, steroids and other medications for treating eye diseases and polysaccharides are incorporated into the present composition, duration of pharmacological action of drugs is extended and side effects are reduced.
When applied in the anterior chamber, the high viscosity of the solution will reduce the aqueous flow and thus hinder corneal endothelium and lens epithelium from obtaining nourishment from the aqueous solution. Similarly, when applied on the external surface of the eye, it hinders corneal epithelium from obtaining nourishments from tears. The presence of .beta.-hydroxybutyrate in the composition overcomes these problems. Namely, .beta.-hydroxybutyrate effectively meets the requirements for the ocular tissues to sustain viability and to perform physiologic functions with concurrent suppression of lactate production and accumulation.
The present isotonic medium is very effective for corneal storage and for preserving corneal endothelium for both humans and animals. Specifically, Fe(NO.sub.3).sub.3, ascorbate and NaHCO.sub.3 are omitted to prevent the formation of free radicals and H.sub.2 O.sub.2 and the shifting of pH from 7.4 to alkaline values when exposed to air. In addition, while the medium is kept isotonic, Cl.sup.- level is reduced from 145 mM to 90 mM and polysaccharides and glucuronate are added to minimize the corneal swelling related to Cl.sup.- passive accumulation and the loss of stromal extracellular matrix substances during storage. When stored in this isotonic medium, stored cornea is are able to perform deturgescence function without resorting to dehydrating agents to maintain the desired thinness.
The specific features of the isotonic medium of the invention are lacking in the prior art. In one method described by Chen, et al. (U.S. Pat. No. 4,873,186), .beta.-hydroxybutyrate is added to Medium 199, which contains NaHCO.sub.3, Fe(NO.sub.3).sub.3 and ascorbate. In another method described by Chen, et al. (divisional application of U.S. Pat. No. 5,116,868), a corneal storage medium is formed by replacing the balanced salt solution of a tissue culture medium with ophthalmic irrigation solution. Both media are hypertonic, up to 370 mOsM, and are effective in storing rabbit corneas for only up to 5 days at 4.degree. C. This is about 50% as effective as the isotonic medium of the present invention. In addition, they do not employ agents such as glucuronate and polysaccharides and a reduced Cl.sub.- level to minimize corneal swelling.
The theory and intended application of polysaccharides and glucuronate for corneal storage are distinctly different from those of dextran and chondroitin sulfate described by Lindstrom, et al. (U.S. Pat. No. 4,695,536 and Am. J. Ophthalmol., 95, 869, 1977) and McCarey, et al. (Invest. Ophthalmol., 13, 165, 1974). These articles describe tissue culture techniques that scientists in the field have been using for years, except for the addition of chondroitin sulfate and/or dextran to exert a colloid osmotic pressure on stored corneas under hypertonic conditions thereby achieving an artificial dehydrating effect. In addition, NaHCO.sub.3, Fe(NO.sub.3).sub.3 and ascorbate are present in these storage media.
It is believed that deturgescence of the cornea is mediated by fluid and ion transports located in the endothelium with energy derived mainly from glucose. Forty-seven percent of the energy is derived from the glycolysis pathway where the pyruvate formed is reduced to lactate at the expense of NADH, an intracellular high energy reserve (Chen, et al., Arch. Biochem. Biophys., 276, 70, 1990). Glycolysis is not generally an energy-efficient pathway, since only 2 moles of ATP are formed per mole of glucose utilized.
It is important to note that lactate is not toxic. In fact, lactate is utilized in vivo in tissues such as the liver and heart. Exogenous lactate is also utilized in the cornea (Kuhlman, et al., Arch. Biochem. Biophys., 85, 29, 1959). However, when pyruvate and lactate are formed in the cells from glucose, two equivalents of H.sup.+ are generated. H.sup.+ produced normally is removed via mechanisms such as facilitated H.sup.+ -lactate symport (Spencer, et al., Biochem. J., 154, 405, 1976) and H.sup.+ /Na.sup.+ antiport (Knicklebein, et al., Am. J. Physiol., 245, 504, 1983), with subsequent clearance, accompanying the lactate through the blood circulation. A clearance mechanism is lacking in tissues under storage and in cell cultures, and is ineffective in tissues under surgery. Under these conditions, glucose metabolism may lead to excessive accumulation of lactic acid outside the cells. This, in turn, inhibits H.sup.+ -lactate symport, which results in cellular acidity with subsequent inhibition of tissue metabolic function (Chen, et al., Arch. Biochem. Biophys., 276, 70, 1990).
Ketone bodies are a collective term for .beta.-hydroxybutyrate (CH.sub.3 CHOHCH.sub.2 COO.sup.-), acetoacetate (CH.sub.3 COCH.sub.2 COO.sup.-) and acetone (CH.sub.3 COCH.sub.3). Ketone bodies, they are readily generated from fatty acids and ketogenic amino acids (such as phenylalanine, leucine, lysine, tryptophan, and tyrosine), with the major metabolic site being in the liver. Among these, acetone is not metabolized and is excreted via breath, sweat and urine. Whereas, acetoacetate and .beta.-hydroxybutyrate are delivered to peripheral tissues for storage or catabolism to generate energy.
Ketone bodies and precursors thereof are efficient energy-rich metabolites, not metabolic wastes. They are synthesized during development (Drahota, et al., Biochem. J., 93, 61, 1964) and in a physiologic response when glucose is inadequate or lacking. For instance, they are elevated in the blood under diabetic conditions and in non-diabetic man following extended fasting. Ketone bodies are the preferred fuel for the brain, muscle, and kidney during starvation (Olson, Nature, 195, 597, 1962; Bassenge, et al., Am. J. Physiol., 208, 162, 1965; Owen, et al., J. Clin. Invest., 46, 1589, 1967; and Howkins, et al., Biochem. J., 122, 13, 1971 and 125 , 541, 1971). They are readily oxidized in peripheral tissues, yielding 32 molecules of ATP per acetyl moiety utilized. In addition, the enhanced respiration will inhibit glycolysis via the Pasteur effect (Krebs, Essays Biochem., 8, 1, 1972), thus reducing lactic acid formation and accumulation.
The NaHCO.sub.3 as high as 25-30 mM is reportedly needed for the cornea to deturgesce in vitro (Hodson, et al., J. Physiol., 263, 563, 1976). However, oxidation of .beta.-hydroxybutyrate is able to generate adequate HCO.sub.3.sup.- for the cornea to perform physiologic functions in vivo normally (Chen, et al., Transplantation, in press, 1994), thus enabling omission of NaHCO.sub.3 from the irrigating solution. This resembles the built-in HCO.sub.3.sup.- -generating system that exists in humans and animals in vivo, in which HCO.sub.3.sup.- is generated from oxidation of ingested foods, not NaHCO.sub.3 contained in the foods.
The present composition without NaHCO.sub.3 has a stable pH and is favorable for clinical applications. When solutions containing NaHCO.sub.3 are used, it requires a calculated P.sub.CO2 to maintain the designated pH's. This can be done in a closed system, such as in vitro experiments. However, in an open system, such as that in surgery and in donor tissue preparation for transplantation, P.sub.CO2 is low and variable. Consequently, the pH of HCO.sub.3.sup.- -containing solutions, such as BSS-plus and McCarey-Kaufman medium, will shift to become alkaline when opened to air, which also causes Ca.sup.2+ to precipitate. According to the manufacturer, BSS-plus has to be discarded after mixing for four hours. It has no mechanisms to prevent precipitation.
The theory and intended application of the present composition differ from the teaching of Veech (U.S. Patent No. 4,663,289). In the teaching of Veech, .beta.-hydroxybutyrate has to be coupled to acetoacetate in a defined ratio, and .beta.-hydroxybutyrate is not intended to use as a high energy source for ocular and peripheral tissues and to inhibit lactate production and accumulation. Veech's invention is based on the theory that the metabolic process in living animal cells can be regulated by a medium containing one or more of the [HCO.sub.3.sup.- ]/[CO.sub.2 ], [.beta.-hydroxybutyrate]/[acetoacetate], and [L-lactate.sup.- ]/[pyruvate.sup.- ] couples in defined ratios. This parallels the buffer system of the "weak acid/conjugated base" couple in regulating pH. Veech's invention is based on several assumptions:
1. Both of the coupled-substrates in a defined ratio in the medium are taken up together by tissues, and remain there at the same ratio to regulate the metabolic process. PA1 2. Uptake and metabolic regulation of these coupled substrates for all tissues are same. PA1 First, all ingredients, except CaCl.sub.2 and MgCl.sub.2, are dissolved and thoroughly mixed in deionized, double-distilled and de-gassed H.sub.2 O, preferably 90%-95% of total volume; and the pH is checked and adjusted to 7.3-7.4 with 1N HCl or NaOH as needed. Second, CaCl.sub.2 and MgCl.sub.2 are added and mixed, followed with H.sub.2 O to make up the volume, and the pH is re-adjusted as needed. Third, the solution is filtered through 0.22 .mu.m membrane (Nalge Co., Rochester, N.Y.), bottled and sealed under vacuum to insure a complete elimination of O.sub.2 from the solution to protect .beta.-hydroxybutyrate from oxidation and to extend the shelf-life. And fourth, the solution is sterilized by autoclave or showers of super-heated water at 121.degree.-123.degree. C. for 15-20 min, and immediately cooled rapidly with showers of water in two stages to prevent breakage of glass bottles, first at 60.degree. C. or between 50.degree. C. and 80.degree. C. depending on quality of bottles used and then at 4.degree. C. until the precipitates disappear.
Much of the experiments, on which Veech based his invention, were done with the rat liver (Veech, et al., Biochem. J., 115, 609, 1969 and J. Biol. Chem., 254, 6538, 1979). In humans and animals, the liver is the metabolic center, and it differs from peripheral tissues in the metabolic process. Therefore, the mechanism for regulating the metabolic process in the liver, may not necessarily work in peripheral tissues. For example, although ketone bodies are synthesized in the liver, they are not utilized there, but rather are translocated to peripheral tissues via the blood circulation. There, they are oxidized to yield a high level of ATP (Principles of Biochemistry, Lehninger, Worth Publishers, Inc.). Thus, .beta.-hydroxybutyrate serve as an efficient fuel for peripheral tissues, but not for the liver.
In addition, the substrate-couples are interchangeable. Whenever one of the metabolites is taken up or produced in the cells, another substrate of the couple is formed. The isozymes of dehydrogenases catalyzing these reactions vary from tissue to tissue. Those in the liver catalyze the reactions towards oxidation; whereas those in peripheral tissues favor reduction. Because of tissue-dependent variations in isozymes and of constant changes in metabolite levels in vivo due to factors such as fluxes, metabolism and food-intakes, it is difficult to establish a generalized ratio for substrate-couples, such as those described by Veech, for all tissues.